Unveiling the Biosynthetic Pathway for Short Mycolic Acids in Nontuberculous Mycobacteria: Mycobacterium smegmatis MSMEG_4301 and Its Ortholog Mycobacterium abscessus MAB_1915 Are Essential for the Synthesis of α'-Mycolic Acids.

Mycolic acids, a hallmark of the genus Mycobacterium, are unique branched long-chain fatty acids produced by a complex biosynthetic pathway. Due to their essentiality and involvement in various aspects of mycobacterial pathogenesis, the synthesis of mycolic acids-and the identification of the enzymes involved-is a valuable target for drug development. Although most of the core pathway is comparable between species, subtle structure differences lead to different structures delineating the mycolic acid repertoire of tuberculous and some nontuberculous mycobacteria. We here report the characterization of an α'-mycolic acid-deficient Mycobacterium smegmatis mutant obtained by chemical mutagenesis. Whole-genome sequencing and bioinformatic analysis identified a premature stop codon in MSMEG_4301, encoding an acyl-CoA synthetase. Orthologs of MSMEG_4301 are present in all mycobacterial species containing α'-mycolic acids. Deletion of the Mycobacterium abscessus ortholog MAB_1915 abrogated synthesis of α'-mycolic acids; likewise, deletion of MSMEG_4301 in an otherwise wild-type M. smegmatis background also caused loss of these short mycolates. IMPORTANCE Mycobacterium abscessus is a nontuberculous mycobacterium responsible for an increasing number of hard-to-treat infections due to the impervious nature of its cell envelope, a natural barrier to several antibiotics. Mycolic acids are key components of that envelope; thus, their synthesis is a valuable target for drug development. Our results identify the first enzyme involved in α'-mycolic acids, a short-chain member of mycolic acids, loss of which greatly affects growth of this opportunistic pathogen.

A Novel Breakthrough in Leptospira spp. Mutagenesis: Knockout by Combination of CRISPR/Cas9 and Non-homologous End-Joining Systems.

Leptospirosis is of general concern as it is a widespread zoonotic disease caused by pathogenic species of the genus Leptospira, although this genus also includes free-living saprophytic strains. Understanding the pathophysiology of leptospirosis is still in its infancy even after several years of its discovery, because of the lack of effective genetic tools. The use of the Streptococcus pyogenes CRISPR/Cas9 system and its variations have pushed the leptospirosis research forward, relying on the simplicity of the technique. However, the lethality of double-strand breaks (DSB) induced by the RNA-guided Cas9 enzyme has limited the generation of knockout mutants. In this work, we demonstrated sustained cell viability after concurrent expression of CRISPR/Cas9 and Mycobacterium tuberculosis non-homologous end-joining components in a single-plasmid strategy in L. biflexa. Scarless mutations resulting in null phenotypes could be observed in most of the colonies recovered, with deletions in the junctional site ranging from 3 to almost 400 bp. After plasmid curing by in vitro passages in a medium without antibiotic, selected marker-free and targeted mutants could be recovered. Knockout mutants for LipL32 protein in the pathogen L. interrogans could be obtained using M. smegmatis NHEJ machinery, with deletions ranging from 10 to 345 bp. In conclusion, we now have a powerful genetic tool for generating scarless and markerless knockout mutants for both saprophytic and pathogenic strains of Leptospira.

A novel breakthrough in Leptospira spp. mutagenesis: knockout by combination of CRISPR/Cas9 and non-homologous end-joining systems

Leptospirosis is of general concern as it is a widespread zoonotic disease caused by pathogenic species of the genus Leptospira, although this genus also includes free-living saprophytic strains. Understanding the pathophysiology of leptospirosis is still in its infancy even after several years of its discovery, because of the lack of effective genetic tools. The use of the Streptococcus pyogenes CRISPR/Cas9 system and its variations have pushed the leptospirosis research forward, relying on the simplicity of the technique. However, the lethality of double-strand breaks (DSB) induced by the RNA-guided Cas9 enzyme has limited the generation of knockout mutants. In this work, we demonstrated sustained cell viability after concurrent expression of CRISPR/Cas9 and Mycobacterium tuberculosis non-homologous end-joining components in a single-plasmid strategy in L. biflexa. Scarless mutations resulting in null phenotypes could be observed in most of the colonies recovered, with deletions in the junctional site ranging from 3 to almost 400 bp. After plasmid curing by in vitro passages in a medium without antibiotic, selected marker-free and targeted mutants could be recovered. Knockout mutants for LipL32 protein in the pathogen L. interrogans could be obtained using M. smegmatis NHEJ machinery, with deletions ranging from 10 to 345 bp. In conclusion, we now have a powerful genetic tool for generating scarless and markerless knockout mutants for both saprophytic and pathogenic strains of Leptospira.

Evaluation of Mycobacterium smegmatis as indicator of the efficacy of high hydrostatic pressure and ultra-high pressure homogenization treatments for pasteurization-like purposes in milk.

The objectives of this study were: to assess the efficiency of high hydrostatic pressure or ultra-high pressure homogenization against Mycobacterium smegmatis in milk and to discuss whether M. smegmatis can be considered a suitable surrogate for other Mycobacterium spp. in high pressure inactivation trials using milk. Three strains of this specie (CECT 3017, 3020 and 3032) were independently inoculated into both skimmed (0.2% fat) and whole milk (3.4% fat) at an approximate load of 6.5 Log CFU/ml and submitted to HHP treatments at 300, 400 or 500 MPa for 10 m at 6°C and 20°C. Evolution of the surviving cells of the inoculated strains was evaluated analysing milk immediately after the treatments and after 5 and 8 d of storage at 6°C. HHP treatments at 300 MPa were seldom efficient at inactivating M. smegmatis strains, but lethality increased with pressure applied in all cases. Generation of sub-lethal injured cells was observed only after 400 MPa treatments since inactivation at 500 MPa was shown to be complete. Significant differences were not observed due to either temperature of treatment or fat content of milk, except for strain CECT3032, which was shown to be the most sensitive to HHP treatments. Milk inoculated with strain CECT3017 was submitted to ultra-high pressure homogenization (UHPH) treatments at 200, 300 and 400 MPa. Maximum reductions were obtained after 300 and 400 MPa treatments, although less than 3.50 Log CFU/ml were inactivated. UHPH did not cause significant number of injured cells. The usefulness of this species as a marker for pressure-based processing seems limited since it showed greater sensitivity than some pathogenic species including other Mycobacteria reported in previous studies.

Single-species (bacterial, fungal, or mycobacterial) biofilms or dual-species (mycobacterial-fungal) biofilms formed in dialysis fluids.

Continuous hemodialysis system monitoring is necessary to prevent microorganism growth and health problems. This study evaluates single- and dual-species biofilm formation in microtiter plates by using dialysis solutions under aerobiosis or 5% CO2 atmosphere. Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Candida parapsilosis sensu lato, and Mycobacterium smegmatis produce single-species biofilms in all dialysis solutions in both oxygenation conditions. Dual-species biofilm cultures grown at 5% CO2 atmosphere and in dialysate containing glucose reveal that M. smegmatis benefits from its association with C. parapsilosis. The dialysate and its constituent solutions support the growth of all the mono-species and the inter-kingdom mycobacterial/yeast biofilms in both aerobiosis and microaerophilic conditions.

The P-type ATPase CtpF is a plasma membrane transporter mediating calcium efflux in Mycobacterium tuberculosis cells.

Among the 12 P-type ATPases encoded by the genome of Mycobacterium tuberculosis (Mtb), CtpF responds to the greatest number of stress conditions, including oxidative stress, hypoxia, and infection. CtpF is the mycobacterial homolog of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) of higher eukaryotes. Its expression is regulated by the global regulator of latency, DosR. However, the role that CtpF plays in the mycobacterial plasma membrane remains unknown. In this study, different functional analyses showed that CtpF is associated with calcium pumping from mycobacterial cells. Specifically, Mtb CtpF expression in Mycobacterium smegmatis cells prevents Ca2+ accumulation compared with wild type (WT) cells. In addition, plasma membrane vesicles from recombinant membranes, in which the direction of ion transport is inverted, accumulate more Ca2+ compared with vesicles obtained from the WT strain. This findings support the hypothesis that CtpF contributes to calcium efflux from mycobacterial cells. Accordingly, Mtb cells defective in ctpF (MtbΔctpF) accumulate more Ca2+ compared with WT cells, while the Ca2+-dependent ATPase activity is significantly lower in the mutant cells. Interestingly, the deletion of ctpF in Mtb impairs the tolerance of the bacteria to oxidative and nitrosative stress. Overall, our results indicate that CtpF is associated with calcium pumping from mycobacterial cells and the response to oxidative stress.

Mutations in the anti-sigma H factor RshA confer resistance to econazole and clotrimazole in Mycobacterium smegmatis.

Azole drugs such as econazole, are active on Mycobacterium tuberculosis and Mycobacterium smegmatis ; however, the identification of their target(s) is still pending. It has been reported that mutations in the non-essential system mmpL5-mmpS5 conferred resistance to econazole in M. tuberculosis . We herein report that an azole-resistant mutant screen in M. smegmatis rendered mutations in rshA, encoding a non-essential anti-sigma H protein.

Deletion of MSMEG_1350 in Mycobacterium smegmatis causes loss of epoxy-mycolic acids, fitness alteration at low temperature and resistance to a set of mycobacteriophages.

Mycobacterium smegmatis is intrinsically resistant to thiacetazone, an anti-tubercular thiourea; however we report here that it causes a mild inhibition in growth in liquid medium. Since mycolic acid biosynthesis was affected, we cloned and expressed Mycobacterium smegmatis mycolic acid methyltransferases, postulated as targets for thiacetazone in other mycobacterial species. During this analysis we identified MSMEG_1350 as the methyltransferase involved in epoxy mycolic acid synthesis since its deletion led to their total loss. Phenotypic characterization of the mutant strain showed colony morphology alterations at all temperatures, reduced growth and a slightly increased susceptibility to SDS, lipophilic and large hydrophilic drugs at 20 °C with little effect at 37 °C. No changes were detected between parental and mutant strains in biofilm formation, sliding motility or sedimentation rate. Intriguingly, we found that several mycobacteriophages severely decreased their ability to form plaques in the mutant strain. Taken together our results prove that, in spite of being a minor component of the mycolic acid pool, epoxy-mycolates are required for a proper assembly and functioning of the cell envelope. Further studies are warranted to decipher the role of epoxy-mycolates in the M. smegmatis cell envelope.

Mycobacterium smegmatis proteoliposome induce protection in a murine progressive pulmonary tuberculosis model.

Tuberculosis (TB) remains an important cause of mortality and morbidity. The TB vaccine, BCG, is not fully protective against the adult form of the disease and is unable to prevent its transmission although it is still useful against severe childhood TB. Hence, the search for new vaccines is of great interest. In a previous study, we have shown that proteoliposomes obtained from Mycobacterium smegmatis (PLMs) induced cross reactive humoral and cellular response against Mycobacterium tuberculosis (Mtb) antigens. With the objective to evaluate the protective capability of PLMs, a murine model of progressive pulmonary TB was used. Animals immunized with PLMs with and without alum (PLMs/PLMsAL respectively) showed protection compared to non-immunized animals. Mice immunized with PLMsAL induced similar protection as that of BCG. Animals immunized with BCG, PLMs and PLMsAL showed a significant decrease in tissue damage (percentage of pneumonic area/lung) compared to non-immunized animals, with a more prominent effect in BCG vaccinated mice. The protective effect of the administration of PLMs in mice supports its future evaluation as experimental vaccine candidate against Mtb.

Characterization of mycobacteria and mycobacteriophages isolated from compost at the São Paulo Zoo Park Foundation in Brazil and creation of the new mycobacteriophage Cluster U.

BACKGROUND: A large collection of sequenced mycobacteriophages capable of infecting a single host strain of Mycobacterium smegmatis shows considerable genomic diversity with dozens of distinctive types (clusters) and extensive variation within those sharing evident nucleotide sequence similarity. Here we profiled the mycobacterial components of a large composting system at the São Paulo zoo. RESULTS: We isolated and sequenced eight mycobacteriophages using Mycobacterium smegmatis mc(2)155 as a host. None of these eight phages infected any of mycobacterial strains isolated from the same materials. The phage isolates span considerable genomic diversity, including two phages (Barriga, Nhonho) related to Subcluster A1 phages, two Cluster B phages (Pops, Subcluster B1; Godines, Subcluster B2), three Subcluster F1 phages (Florinda, Girafales, and Quico), and Madruga, a relative of phage Patience with which it constitutes the new Cluster U. Interestingly, the two Subcluster A1 phages and the three Subcluster F1 phages have genomic relationships indicating relatively recent evolution within a geographically isolated niche in the composting system. CONCLUSIONS: We predict that composting systems such as those used to obtain these mycobacteriophages will be a rich source for the isolation of additional phages that will expand our view of bacteriophage diversity and evolution.

Pma1 is an alkali/alkaline earth metal cation ATPase that preferentially transports Na(+) and K(+) across the Mycobacterium smegmatis plasma membrane.

Mycobacterium smegmatis Pma1 is the orthologue of M. tuberculosis P-type ATPase cation transporter CtpF, which is activated under stress conditions, such as hypoxia, starvation and response to antituberculous and toxic substances. The function of Pma1 in the mycobacterial processes across the plasma membrane has not been characterised. In this work, bioinformatic analyses revealed that Pma1 likely contains potential sites for, Na(+), K(+) and Ca(2+) binding and transport. Accordingly, RT-qPCR experiments showed that M. smegmatis pma1 transcription is stimulated by sub-lethal doses of Na(+), K(+) and Ca(2+); in addition, the ATPase activity of plasma membrane vesicles in recombinant Pma1-expressing M. smegmatis cells is stimulated by treatment with these cations. In contrast, M. smegmatis cells homologously expressing Pma1 displayed tolerance to high doses of Na(+) and K(+) but not to Ca(2+) ions. Consistently, the recombinant protein Km embedded in plasma membrane demonstrated that Ca(2+) has more affinity for Pma1 than Na(+) and K(+) ions; furthermore, the estimation of Vmax/Km suggests that Na(+) and K(+) ions are more efficiently translocated than Ca(2+). Thus, these results strongly suggest that Pma1 is a promiscuous alkali/alkaline earth cation ATPase that preferentially transports Na(+) and/or K(+) across the mycobacterial plasma membrane.

Mycobacterium smegmatis synthesizes in vitro androgens and estrogens from different steroid precursors.

Fast-growing mycobacteria such as Mycobacterium sp. and Mycobacterium smegmatis degrade natural sterols. They are a model to study tuberculosis. Interestingly, M. smegmatis has been found in river effluents derived from paper production, and therefore, it would be important to gain further insight into its capacity to synthesize steroids that are potential endocrine disruptors affecting the development and reproduction of fishes. To our knowledge, the capacity of M. smegmatis to synthesize estrogens and even testosterone has not been previously reported. Therefore, the objective of this study was to investigate the capacity of M. smegmatis to synthesize in vitro testosterone and estrogens from tritiated precursors and to investigate the metabolic pathways involved. Results obtained by thin-layer chromatography showed that (3)H-progesterone was transformed to 17OH-progesterone, androstenedione, testosterone, estrone, and estradiol after 6, 12, or 24 h of incubation. (3)H-androstenedione was transformed into testosterone and estrogens, mainly estrone, and (3)H-testosterone was transformed to estrone and androstenedione. Incubation with (3)H-dehydroepiandrosterone rendered androstenediol, testosterone, and estrogens. This ability to transform less potent sex steroids like androstenedione and estrone into other more active steroids like testosterone and estradiol or vice versa suggests that M. smegmatis can influence the amount of self-synthesized strong androgens and estrogens and can transform those found in the environment.

Evaluación del potencial de Mycobacterium smegmatis como candidato vacunal contra la tuberculosis mediante estudios in sílico e in vivo / Evaluation of the potential of Mycobacterium smegmatis as vaccine Candidate against tuberculosis by in silico and in vivo studies

In this study, we scanned multiple published databases of gene expression in vivo of M tuberculosis at different phases of infection in animals and humans, to select 38 proteins that are highly expressed in the active, latent and reactivation phases. The selected proteins were predicted for T and B epitopes. For each proteins, the regions containing T and B epitopes were selected at the same time to look for identical epitopes on M smegmatis based on sequence alignments. Preliminary studies of humoral immunogenicity and cross-reactivity with M tuberculosis in mice using two M smegmatis-derived experimental vaccines were carried out, demonstrating the immunogenicity of M smegmatis proteoliposomes and the recognition of M tuberculosis proteins by the sera of animals immunized with this vaccine candidate. The conjunction of in silico and in vivo studies suggested the potential for future evaluation of M smegmatis as vaccine candidate against tuberculosis using different strategies.(AU)

En este estudio se revisaron múltiples bases de datos publicadas, relacionadas con experimentos de expresión de genes de M tuberculosis in vivo en diferentes estadios de la infeccción en humanos y animales. Se identificaron 38 proteínas con elevada expresión en las fases activa, latente y de reactivación de la infección. Se llevó a cabo la predicción de epítopes T y B en dichas proteínas. Las regiones de cada proteína que contenían simultàneamente epítopes T y B se seleccionaron y utilizaron para identificar regiones idénticas en M smegmatis mediante el alineamiento de secuencias. Se llevaron a cabo estudios de inmunogenicidad humoral y reactividad cruzada con M tuberculosis en ratones inmunizados con dos vacunas experimentales obtenidas a partir de M smegmatis, demostràndose la immunogenicidad de los proteoliposomas y el reconocimiento de proteínas de M tuberculosis por el suero de ratones vacunados con este candidato vacunal. Los resultados obtenidos con los estudios in sílico e in vivo sugieren la potencialidad para evaluación futura de candidatos vacunales obtenidos a partir de M smegmatis para la prevención de la tuberculosis(AU)

Obtención y caracterización parcial de un extracto lipídico de la membrana externa de Mycobacterium smegmatis / Obtainment and partial characterization of a lipidic fragment of the outer membrane of Mycobacterium smegmatis

En la actualidad, los antígenos lipídicos de las micobacterias constituyen blancos atractivos para el desarrollo de nuevas formulaciones vacunales contra la tuberculosis. En nuestro trabajo se realizó la caracterización parcial de un extracto lipídico de pared celular de Mycobacterium smegmatis mediante cromatografía de capa delgada y Dot blot frente a gammaglobulina humana. Se identificó, fundamentalmente, la presencia de fosfolípidos y ßcidos micólicos en el extracto lipídico y se observó un elevado reconocimiento de los mismos por la gammaglobulina humana, lo cual indica la importancia de continuar los estudios de inmunoprotección empleando antígenos lipídicos de micobacterias(AU)

Currently, lipid antigens of mycobacteria are attractive targets for the development of new tuberculosis vaccinal formulations. A lipid extract of Mycobacterium smegmatis cell wall was characterized using a Thin Layer Chromatography and Dot blot with human gammaglobulin. Mainly we identified the presence of phospholipids and micolic acids in the lipid extract showing a high recognition by human gammaglobulin. These results indicate the relevance of continuing immunoprotection studies with mycobacterial lipid antigens(AU)